The escalating global challenges of food security, waste management, and environmental sustainability have necessitated the application of novel green approaches to utilize agro-industrial food waste as a valuable bioresource. Enzymatic conversion of food waste into high-value bioproducts emerges as one of the promising solutions to address these issues. The current review explores the critical role of enzymes in facilitating the conversion of diverse agro-industrial residues, including those from plants and animals, into biochemicals and functional ingredients. The current review evaluates the environmental and economic benefits of enzyme-mediated bioconversion processes, underlining the circular economy paradigm, which prioritizes resource efficiency and waste minimization. The enzyme production from diverse plant and animal-based food wastes as raw materials has been highlighted beside the description of different bioproducts from food waste using enzymes and the integration of enzymatic bioprocessing. The economic analysis and case studies of the enzyme-mediated processes for biochemical production from food waste have also been emphasized. By harnessing the synergistic potential of enzymes and circular economy principles, the bioconversion of agro-industrial food waste into novel bioproducts presents a viable pathway toward sustainable resource utilization and a circular bioeconomy.
Probiotic foods, generally dairy-based, aren’t widely affordable in low-income countries. So it’s necessary to suggest suitable and accessible matrices for delivering probiotics.
This study aimed to assess the probiotic traits of Lactiplantibacillus plantarum LO3 and its use in the development of a golden apple-based non-dairy probiotic beverage.
To this end, the probiotic and safety properties of Lact. plantarum LO3 were evaluated. Then, Lact. plantarum LO3 suspension was added to GaJ (≈ log 7.67 cfu) and stored at 4 and 30 °C. During storage, the proximate composition, the DPPH° activity as well as a sensory evaluation of the juice were performed.
As a result, Lact. plantarum LO3 has excellent viability (˃97%) in gastric and intestinal juices respectively, after 2 and 4 h. As for adhesive properties, the highest co-aggregations were recorded against Escherichia coli (23.43%) and Vibrio parahaemolyticus (22.05%). In the GaJ, except day 21, Lact. plantarum LO3 load was significantly (p˂0.05) higher than the initial load. Ascorbic acid content decreased over time, with minima recorded on day 30 (22.41 and 25.00 mg/100 ml) at 30 and 4 °C respectively. Furthermore, the highest DPPH° activities (EC50) were 90.05 and 94.56 µg/ml at 4 and 30 °C respectively. Carbohydrates and fibre contents decreased significantly (p˂0.05) with storage temperature. In terms of sensory attributes, Lact. plantarum LO3 had a positive effect on odour at 30 °C, while colour was better preserved at 4 °C.
This makes golden apple juice a suitable matrix for carrying the probiotic strain Lact. plantarum LO3 for consumers from the whole spectrum of social classes.
Nitrile hydratase (NHase) is a metalloenzyme that catalyzes the conversion of nitrile to amide and is widely used in the biocatalysis of bulk chemicals such as acrylamide and nicotinamide. Improving the thermostability, activity, and soluble expression of natural NHase is crucial for its industrial application. However, conventional engineering strategies are often based on the design and evaluation of single-point mutations, followed by multiple rounds of iterative combinations, which are inefficient and difficult to predict the evolutionary direction of the combinatorial mutations due to epistatic effects. In this study, we used PROSS, an automated design tool based on structural and sequence information, to design a thermophilic NHase from Pseudonocardia thermophila JCM3095 (PtNHase). By sequentially applying subunit-independent mutations, subunit-synergistic mutations, and single-point revertant mutations, we obtained the superior mutant A2B1–β221. This mutant exhibited 1.4-fold and 2.3-fold higher activity towards acrylonitrile and 3-cyanopyridine, respectively, compared to the wild type. Additionally, A2B1–β221 showed a significant enhancement in thermostability. Moreover, benefiting from the enhanced soluble expression, a high-performance whole-cell catalyst for NHase was obtained. Furthermore, conventional molecular dynamics simulations and metadynamics simulations were employed to resolve the molecular mechanisms underlying the high activity and thermostability of A2B1–β221. This study not only provided highly efficient whole-cell catalyst for NHase, but also demonstrated the efficacy of utilizing automated design tools and molecular dynamics simulations in the engineering of heterologous multimeric proteins, offering valuable insights into their applicability.
UDP-sugars, as active forms of monosaccharides, play integral roles in glycosylation and biosynthesis of polysaccharides. Although enzymatic catalysis has achieved great process, the comparatively low productivity and the time-consuming enzyme purification processes restricted its practical applications. Here, we developed two CipA-dependent enzyme immobilization systems for synthesis of UDP-GlcNAc and UDP-GlcA. Initially, we selected and identified the enzyme combinations of PpAmgK and SeGlmU for UDP-GlcNAc (3.19 mM) synthesis, and AtGlcAK and BlUSP for UDP-GlcA (1.83 mM) synthesis. After optimizing the molar ratios of substrates, the production of UDP-GlcNAc and UDP-GlcA increased to 17.33 mM and 9.03 mM when setting UTP:GlcNAc:ATP and UTP:GlcA:ATP as 1:1:1 and 1:2:1, respectively. Then, the polyphosphokinase SePPK for recycling ADP and PPi was introduced, resulting in a significant increase in UDP-GlcNAc (29.33 mM) and UDP-GlcA (20.87 mM). Eventually, the CipA-based immobilization systems were developed for repetitive catalysis. The combinations of PSK-(G4S)3-CipA and CipA-(G4S)3-ABK yielded the comparable productions of UDP-GlcNAc (28.66 mM, 17.40 g/L) and UDP-GlcA (20.34 mM, 11.80 g/L) within 75 min. This study presents a convenient and reusable CipA-based enzyme immobilization system for synthesis of UDP-sugars, showing great potential for enzymatic production of UDP-GlcNAc and UDP-GlcA.
Sustainable cultivation strategies is a prerequisite for algal biorefineries targeting on reducing water and energy footprint. Thus, in this study, spent waters from the air conditioning unit (SP1), the water purifier/RO unit (SP2) and from the condenser tube of distillation unit (SP3) was filtered, autoclaved and reused in combination with artificial seawater- f/2 (ASW) media for cultivating oleaginous diatoms, Chaetoceros gracilis and Thalassiosira weissflogii and examine its impact on the growth and lipid production. Both strains showed a sharp rise in cell numbers in the test culture setups supplemented with 50% SP1 than in the control. Biomass productivity and total lipid content was highest in 50% SP1 cultures of C.gracilis (0.045 g L−1 d−1; 14.8% DW) and in 100% SP2 and 100% SP3 culture of T.weissflogii (0.06 g L−1 d−1; 19.6% DW), respectively. Indeed, the results validate for the first time the strategy of recycling spent waters recovered from various laboratory and industrial appliances as an optimized media for cultivating diatom algae via a carbon neutral and cost-effective approach.
Gamma-aminobutyric acid is a versatile and non-protein amino acid that plays a significant role in medicine, food, and cosmetics. The synthesis of gamma-aminobutyric acid is restricted by complex metabolic mechanisms and suboptimal fermentation conditions. Previously, we had constructed the Corynebacterium glutamicum strain CGY-PG-304 which could efficiently produce gamma-aminobutyric acid. In this study, we promoted gamma-aminobutyric acid production in CGY-PG-304 by enhancing the carbon flow in the TCA cycle, streamlining the mycolic acid layer of the cell wall, and optimizing the fermentation conditions. First, the genes sucCD encoding succinyl coenzyme A synthase, the gene cmrA encoding the ketoacyl reductase, and the gene treY encoding maltooligosaccharyl trehalose synthase were deleted in CGY-PG-304 individually or in combination. The yield of gamma-aminobutyric acid was increased in all the resulting strains among which CGW003 was the best. Next, the gene acnA encoding cis-aconitase or the gltS encoding sodium-coupled glutamate secondary uptake system were overexpressed in CGW003 using plasmid, and the former produced more gamma-aminobutyric acid than the latter. Therefore, the promoter of the chromosomal gene acnA in CGW003 was replaced by the strong promoter PtacM, resulting in the final strain CGW005. CGW005 could produce 112.03 g/L of gamma-aminobutyric acid with a yield of 0.34 g/g of glucose by fed-batch fermentation.
To improve the inhibitory insensitivity towards Aspergillus usamii E001 GH11F xylanase (Auxyn11A) towards xylanase inhibitor protein (XIP-I), and Triticum aestivum xylanase inhibitor (TAXI-I) the site-directed mutagenesis was conducted based on the computer-aided redesign. Firstly, two xylanase inhibitory proteins-encoding xip-I and taxi-I were synthesized and expressed in Pichia pastoris SMD1168, namely SyXIP-I and SyTAXI-I. The highest inhibitory activities of SyXIP-I and SyTAXI-I on Auxyn11A occurred at molar ratio (xylanase:inhibitor) 10:1, 40 °C for 30 min, and 100:1, 30 °C for 30 min, respectively. Secondly, nine variants of a Auxyn11A-encoding gene (Auxyn11A) were constructed as designed theoretically and expressed in E. coli BL21(DE3), respectively. One mutant, Auxyn11ASDSS exhibited the highest xylanase activity and showed 100% resistance to the inhibitor SyXIP-I, and Auxyn11AN2 showed 30% resistance. Secondly, to obtain both types of resistance, four variants, Auxyn11AN2−SDSSA, Auxyn11ASDSSA, AuxynMSDSS, and AuxynMSDSSA was also constructed. Among them, AuxynMSDSSA exhibited complete resistance to the SyXIP-I and SyTAXI-I. Furthermore, AuxynMSDSSA was purified. Its optimum temperature and pH were 60 °C and 5.0, and those of Auxyn11A were 50 °C and 5.0. The specific activity, K m, V max, and k cat/K m values of AuxynMSDSSA were 1054.6 U/mg, 4.0 mg/mL, 6659.6 µmol/min/mg, and 610.46 mL/mg/s, which were similia to those of Auxyn11A, 1097.4 U/mg, 4.23 mg/mL, 6876.5 µmol/min/mg, and 557.04 mL/mg/s, respectively. This study provided a novel thermophilic xylanase with complete resistance to the SyXIP-I and SyTAXI-I, therefore, making it a promising candidate for extensive applications in animal feed and food industry.
The semi-rational design of enzymes has become a popular and effective modification method to improve their hydrolytic activity and/or thermal stability toward target substrates. Here, the specific activity of a maltogenic amylase from Lactobacillus rhamnosus YXY412 (LrMA) toward soluble starch was exactly enhanced through hotspot-based research. Based on multiple sequence alignment, three-dimensional structure and existed literature, thirty-eight amino acid residues of LrMA were rationally selected for site-directed mutagenesis. After the screening of the mutants, LrMAD172A, LrMAG260A, LrMAK334A and LrMAM477A were selected with the activity accounted for 144–209% of that in wild-type. Among all the mutants, LrMAG260A possessed the highest activity toward soluble starch, reached 133 U/mg, about twice as high as that in the wild-type. Its temperature for optimum activity still maintained at 60 °C, while had no significant loss of thermal stability occurred. In addition, compared with the wild-type in pH stability, the mutant retained over 80% residual activity at a wider pH range of 4.5–8.5. Furthermore, the k cat/K m of LrMAG260A was two times higher than that of the wild-type, indicating that the mutant had a better affinity and a higher conversion efficiency for soluble starch.
Bacillus licheniformis is an industrially significant microorganism known for its broad carbon source utilization compared to other bacteria. However, the mechanisms underlying this utilization are tightly controlled by global regulatory proteins such as CodY, and the details of these mechanisms remain elusive. This poses challenges for metabolic engineering efforts. In this study, we used the urease encoding gene as a reporter to establish a CRISPRi system based on conditional dCas9 expression in B. licheniformis. The induction with mannose resulted in an 84% transcriptional inhibition of ureA, and a 57% reduction in urease activity, confirming the system's successful construction. We designed three different sgRNA sites within the 5'-end coding region of the codY gene to achieve varying degrees of protein expression knockdown. The results showed that a 10–75% knockdown of codY led to a 23–87% decrease in the maximum specific uptake rates of glucose and maltose. Concurrently, the accumulation of carbon overflow metabolites such as 2,3-butanediol (2,3-BDO) and acetate decreased by 38% and 26%, respectively. These findings enhance our understanding of CodY’s regulatory role in catabolism and metabolism. The CRISPRi system with conditional dCas9 expression developed here serves as an effective synthetic biology tool for metabolic pathway engineering.
Lacto-N-neotetraose (LNnT), with promising bioactive properties, is one of the most significant nonfucosylated human milk saccharides. Recently, its synthesis by microbial fermentation has attracted great attention. However, most recent studies have focused on the use of plasmid for high-level LNnT production, which is undesirable for industrial applications. Therefore, this study aimed to construct a plasmid-free recombinant strain to biosynthesize LNnT. First, a de novo synthesis pathway for LNnT was constructed in Escherichia coli. Then, the ribosome binding site (RBS) and expression pattern of β-1,3-N-acetylglucosaminyltransferase (LgtA) and β-1,4-galactosyltransferase (LgtB) were optimized. Different fusion peptides and enzyme assembly scaffolds were also verified for LNnT enhancement, which resulted in an LNnT titer of 1.70 g/L. Furthermore, a clustered regularly interspaced short palindromic repeat-mediated interference (CRISPRi) system was introduced to downregulate the competitive pathway, thereby enhancing the supply of two precursors (UDP-N-acetylglucosamine and UDP-galactose). Finally, a plasmid-free engineered strain was developed via genome integration of lgtA and lgtB coupled with enzyme assembly scaffolds, which produced 23.73 g/L of LNnT in a 3-L bioreactor. These findings provide insights into the plasmid-free recombinant E. coli strain construction for the efficient LNnT biosynthesis.
Microalgal biorefineries have emerged as significant reservoirs of therapeutic compounds, including pigments and proteins. Facilitating a robust circular bioeconomy necessitates the augmentation of pigment synthesis alongside algae biofuel production. Nevertheless, inherent constraints in ketocarotenoid synthesis exist in naturally fast-growing microalgae strains, such as Chlamydomonas reinhardtii. To address this limitation, we overexpressed two pivotal enzymes in the carotenoid biosynthetic pathway, namely β-carotene hydroxylase (crt) and β-carotene ketolase (bkt), in C. reinhardtii utilizing strong promoters to amplify carotenoid production. The genetically modified (GM) microalgae were validated through PCR, Southern hybridization, and Western blot assays, confirming the presence and expression of both genes in the C. reinhardtii strains. These GM lines exhibited a substantial enhancement over wild-type (WT) algae, showcasing a remarkable 5.39-fold increase in β-carotene concentration and twofold increase in total carotenoids compared to the WT microalgae. Notably, the GM microalgae achieved astaxanthin production up to 1.47 ± 0.063 mg/g DCW, a compound absent in WT C. reinhardtii. These findings indicate the successful functionalization of Hematococcus pluvialis genes through nuclear expression in C. reinhardtii, facilitating ketocarotenoid production. This study presents a valuable strategy to boost carotenoid production in microalgae by stable overexpression of two heterologous genes within the nuclear genome of C. reinhardtii.
Graphical abstract for the study carried out which represents the in silico plasmid vector designing, algae transformation by electroporation, selection on antibiotic plates, PCR amplification for GM confirmation, Southern hybridization to confirm gene integration, Western blotting to check protein expression, pigment quantification, and algae growth determination.
Fluorescent protein optimized for promoter fluorescence intensity in S. cerevisiae.
Short promoter library with different strengths and stable expression constructed.
These promoters express ovalbumin fusion fluorescent proteins in S. cerevisiae.
3-O-Deacyl-2-O-palmitoyl-4′-monophosphoryl lipid A (MPL) has recently been used in vaccine adjuvant. In this study, an E. coli mutant WZM012 which can effectively produce MPL has been constructed from E. coli MG1655 by genome editing. First, the genes mlaE, pldA, and hns related to the phospholipid transport system in membrane were deleted in MG1655 to accumulate more phospholipid substrate for PagP. In addition, the gene FnlpxE from Francisella novicida which can remove the phosphate group at 1-position of lipid A and the gene SepagL from Salmonella which can remove the acyl chain at 3-position of lipid A were inserted into the chromosome to replace the gene clusters ybgC-cpoB and letAB, respectively, resulting in the strain WZM012. After 24 h fed-batch fermentation of WZM012, lipid A species were isolated and analyzed using thin-layer chromatography and liquid chromatography–mass spectrometry, and 32.76 mg/L MPL was obtained. This engineered E. coli strain could be developed for industrial MPL production.
The tolerance of Lactiplantibacillus plantarum (L. plantarum) to iso-α-acid is critical to sour beer production, and the mechanism of its tolerance to iso-α-acid on cell phenotype is still uncovered. In current study, the tolerance of L. plantarum J6 was enhanced by adaptive laboratory evolution (ALE). And the stress response of L. plantarum J6-6 was analyzed by the changes of intracellular protein, cell membrane fatty acid composition and permeability. The strain J6-6 with iso-α-acid (25 mg/L) displayed an increased unsaturation of fatty acids. The protein involved in glycolysis pathway, nucleotide, amino acid and energy metabolism, would enhance the tolerance of J6-6. In addition, ALE can reduce the damage and improve the viability of iso-α-acid. The application of J6-6 in sour beer can easily control the entire fermentation, with more sweet-sour taste and better flavor. This study provides a well-defined target of easy-to-control sour beer.
Maltotetraose (G4) consists of four glucose units linked by an α-1,4-glycosidic bond. This compound demonstrates remarkable versatility in food processing and exhibits specific physiological functions, suggesting promising applications in the medical, chemical, and food sectors. However, due to the closely related physical and chemical properties of maltotriose (G3), G4, and maltopentose (G5), achieving high-purity G4 has been challenging, resulting in a staggering price of US$438.88 per gram. In this study, a novel and efficient bio-physical method was developed to produce high-purity G4. Initially, multi-enzymatic hydrolysis yielded G4 at a 65.83% purity. Subsequent processes involving yeast fermentation and SMB separation further enhanced the purity to an impressive 93.15%. Notably, this pioneering method represents the successful separation of G3, G4, and G5 to exclusively obtain high-purity G4 from maltooligosaccharides, surpassing previous purity achievements. Every facet of this bio-physical method underwent meticulous design and optimization, ensuring a production process that is environmentally friendly, safe, and efficient. To validate its practicality, pilot-scale production tests were conducted. The cost analysis indicates that producing high-purity G4 through this method amounts to only US$0.013 per gram, representing that the actual selling price of G4 was 33,760 times the production cost under this process.
Escherichia coli, a Gram-negative bacteria is extensively utilized as a microbial platform for the recombinant protein production. Various modifications have been introduced to E. coli strains to maximise the production of different types of recombinant proteins. However, a limited amount of research is dedicated to the manipulation of the central carbon metabolic pathways of E. coli. The synthesis of proteins relies on the utilization of carbon and energy precursors from the central carbon metabolic pathways. The high demand for precursors and energy in the biosynthesis of heterologous proteins prompts the cells to readjust their anabolic and catabolic reactions. This readjustment gives rise to another issue, namely the acetate accumulation, which leads to a decline in biomass and production of recombinant protein. Therefore, an improved engineering strategy would involve the manipulation of global regulators and gene sets, encompassing multiple pathways, to enhance carbon assimilation while avoiding the formation of by-products such as acetate. Thus, a synergistic approach has been proposed to boost the productivity of recombinant L-Asparaginase-II, which involves the co-expression of the Catabolite repressor activator (cra) gene, a global regulator of central carbon metabolism, and the deletion of acetate biosynthesis genes. This approach has demonstrated a significant increase in the yield of L-Asparaginase-II in both E. coli BW25113ΔpoxB and BW25113Δpta cultures, where cra is co-expressed using a plasmid-based system. A pelB signal sequence leads to extracellular secretion of recombinant L-Asparaginase-II and it was observed that the productivity of E. coli BW25113ΔpoxB and BW25113Δpta cultures was 129% and 66% higher than wild type BW25113 strains.
Environmental issues such as air pollution and climate change due to excessive fossil fuel burning; plastic pollution and wastewater contamination are a cause of global concern. Bacteria have the ability to not only utilize organic contaminants present in wastewater, but also to synthesize bioproducts such as fatty acid methyl esters (FAMEs), the primary molecules in biodiesel and Polyhydroxyalkanoates (PHAs) or bioplastics. The present study aims to investigate production of FAMEs and PHA along with biodegradation of wastewater contaminants by sewage sludge bacteria. Screening of sewage sludge bacteria was done for their lipid/PHA accumulation potential in filter sterilized wastewater by viable colony, fluorescence microscopy and spectrofluorometry methods using nile red staining. For characterization of bacterial FAME and PHA, cultures were subjected to in situ transesterification and analysed using gas chromatography mass spectrometry (GC-MS). A previously reported thermotolerant bacterial strain Bacillus sp. ISTVK1, that showed promising results was further tested for its the potential to produce FAME/PHA along with wastewater contaminant removal by performing physico-chemical analyses, scanning electron microscopy-energy dispersive X-ray (SEM-EDX) spectroscopy and transmission electron microscopy (TEM). The analysis showed the presence of C15 − 18 FAMEs such as Hexadecanoic acid, methyl ester and Tetradecanoic acid 12-methyl ester and PHA such as Pentanoic acid, 4-oxo, methyl ester. Physico-chemical analysis of culture supernatant revealed a 92% reduction in COD and the absence of contaminants such as Benzene, 1,1’-(2-pentene-1,5-diyl)bis (R.T.= 25.42) and 5-(1-Phenyl-cyclopentyl)- [1,3,4]oxadiazole-(R.T.=27.75) from post-treated samples. Results of SEM-EDX and TEM further confirmed bacterial lipid/PHA accumulation and contaminant reduction post-treatment. The potential of the thermotolerant Bacillus sp. ISTVK1 for biodiesel and biopolymer production along with wastewater contaminant removal, as revealed in this study could have future implications in various thermal and polymer industries and wastewater valorization technologies.
About 39 million tons of Brewer’s spent grain (BSG), the main by-product of beer manufacturing, are produced annually and is used for low value applications. To valorise this rich bioresource, the present work entails bioprocessing of BSG with various proteases (Novozymes) at two different concentrations (4% and 9%) to solubilise protein, carbohydrate and polyphenols from the grain and evaluate its effect on gut and brain health. The results show the highest (p < 0.05) FRAP (Ferric Reducing Antioxidant Power) based antioxidant activity was obtained for Pro 5 (4%: 108.10 ± 4.17 µmole Trolox Equivalence (TE)/g protein, 18.06 ± 0.70 µmole TE/g d.w., which was 3.6 times higher than untreated control extracts. The highest DPPH (1,1-diphenyl-2-picrylhydrazyl) scavenging activity was obtained for the same extract (Pro 5 at 4%: 0.118 ± 0.006 µg AAE/mg d.w., 9%: 0.110 ± 0.006 µg AAE/mg d.w). Proximate composition showed this extract to contain the highest concentration of proteins at 21.66% ± 2.71, and color analysis showed the same Pro 5 (4%) extract to be the darkest (L*53.73) indicating the possible presence of dark polyphenols. Anticancer screening showed Control and Pro 1 to possess cytotoxic effect against colon cancer cells with IC50 of 3.2 and 13.91 mg/mL, respectively. No significant activity was noted against the brain cancer cell line. Thus anti-colon cancer activity of BSG extracts highlights its potential in gut health. The observed bioactivity resulted from a combination of peptides, carbohydrates and polyphenolic compounds in the extract and warrants further characterisation for targeted nutraceutical applications.
The influence of hydrogen-rich water (HRW) on the antioxidant activity and its inherent mechanism were investigated during barley malting. The antioxidant activity was evaluated with 2,2-diphenyl-1-picryhydrazyl (DPPH) radical scavenging activity, 2,2’-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) radical cation scavenging activity, reducing power, and metal chelating activity. Our results revealed that HRW (1.2 mg/L) significantly increased the antioxidant activity of malt, which was supported by the finding that HRW could effectively promote the synthesis of phenolic substances. Furthermore, HRW treatment enhanced the activities of catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) and decreased the activity of polyphenol oxidase (PPO) in malt. Thus, our research demonstrated that HRW can be used to produce malt to increase its antioxidant activity effectively.
γ-Aminobutyric acid (GABA) is a bioactive compound with diverse physiological functions. It has a wide range of applications in food and medicine and mainly biosynthesized through glutamate decarboxylase (GAD) catalysis. Bacillus subtilis is recognized for its robust secretion capabilities and high food safety standards, making it a prevalent choice for recombinant protein expression. In traditional industrial enzyme production in B. subtilis, antibiotics are required to sustain plasmid stability. However, the incorporation of antibiotics fails to align with the criteria applicable to enzymes for use in the food industry. To eliminate the need for antibiotics in the production of GAD preparations, we constructed a marker-free recombinant strain B. subtilis WS9D-GAD. This strain was developed based on antibiotic-free multi-copy gene expression vector pUBDAL-amyL and d-alanine racemase (dal)—deficient B. subtilis WS9D, ultimately enabling the food-grade expression of GAD. To enhance GAD expression levels, we integrated the gadA expression cassette into the genome of B. subtilis using the Cre/lox system method. Additionally, strain WS9C6D-GAD was generated through the co-expression of free plasmid and genome integration, which carried the free plasmid pUBDAL-gadA and featured six copies of the gadA expression cassette within its genome. The enzyme activity during shake flask fermentation reached 28.15 U mL−1, while the enzyme activity in high-density 3-L fermenter culture reached 199.49 U mL−1, marking the highest level of food-grade GAD expression with a multitude of potential applications. This study presents an effective strategy for the expression of food-grade industrial enzymes in B. subtilis.
Petroleum-based plastics have been associated with several environmental issues, including land and water pollution, greenhouse gas emissions, and waste accumulation due to their non-biodegradable properties. Bioplastics derived from renewable natural resources have emerged as an eco-friendly substitute for conventional plastics, leading to a reduced carbon footprint and conservation of non-renewable fossil fuels. Seaweed is an attractive material for bioplastic production due to its abundant polysaccharide content, high biomass, rapid growth rate and suitability for consumption. This work aimed to explore the feasibility of producing seaweed bioplastics, specifically starch and carrageenan from Kappaphycus alvarezii, along with chitin extracted from ramshorn snails (Planorbarius corneus). The surface morphology of the bioplastics was assessed through scanning electron microscopy (SEM), and their biodegradability was also examined through a soil burial biodegradation test. Starch-based bioplastics incorporated with carrageenan and chitin exhibited a more substantial network structure, rougher surface texture and smaller void sizes with improved mechanical strength and water barrier properties. The bioplastics underwent decomposition, resulting in fragmentation into small pieces (with more than 76% weight loss) or complete degradation through the enzymatic activity of Acinetobacter spp. and Burkholderia cepacia. Therefore, seaweed-chitin-based bioplastics demonstrate their potential as a sustainable and environmentally friendly alternative to conventional plastics.
Violacein, a therapeutic pigment synthesised naturally in specific bacterial systems, is commercially unexplored owing to low titers. The current study aimed to formulate a suitable medium and to develop a fed-batch strategy for improving the violacein productivity in a natural producer—Chromobacterium violaceum MTCC2656. The carbon and the nitrogen sources were extensively screened and their levels were optimised for maximal violacein production. The micronutrients in the medium were subjected to a two-level statistical optimisation using design of experiment approach and the final media formulation was validated. As a fed-batch approach, a combination of pulse feeding of glucose and tryptophan with optimised broth harvest of 60% (v/v) was attempted that achieved a titer of 1046 ± 16 mg/L with productivity of 26.12 ± 0.64 mg/L/h in each progressive cycle of fed-batch. The strategic sequential step of media formulation and fed-batch fermentation improved the violacein titer by ~ 5 folds. Kinetic modelling was used to understand the enhancement in fermentation performance in both the batch and fed-batch processes. The findings from the current study would enable to understand and correlate the patterns of substrate uptake and violacein formation to establish strategies for enhancing overall productivity in the fermentation processes.
Bacillus subtilis, as a model microorganism with a clear background, has the advantages of strong secretion ability and a generally recognized as safe status. Although the production of heterologous proteins is increasing with the development of biotechnology, the expression level of many heterologous proteins could not meet the requirements for industrial application. Here, to further enhance the production of α-amylase from Bacillus stearothermophilus (AmyS) which is industrially important due to its wide application, 12 potential expression-related genes were selected due to upregulation in high production strain and, respectively, overexpressed to evaluate their function in the expression of AmyS in B. subtilis 1A976. The highest enzyme activity was obtained by overexpression of ponA (1.58-fold), which was the major penicillin-binding protein in cell wall synthesis. In addition, the sources and expression level of ponA were investigated. Subsequently, to exert the superb secretion ability of B. subtilis WS9, a host strain with excellent expression capability, these identified enhancers were, respectively, investigated in this strain. Due to the extremely low transformation efficiency of B. subtilis WS9, many attempts were taken to improve transformation efficiency of B. subtilis WS9. As a decisive regulator of the competent cell formation, comK was integrated into the genome of B. subtilis WS9, named as WS9C. The easy-transformable WS9C highly facilitated the subsequent genetic manipulation. Integration of ponA also increased the production of AmyS (1.37-fold) in WS9C. On this basis, combinatorial overexpression of ponA with other five screened genes liaH, oppA, secA, prsA, and ltaS was performed, respectively, and the most suitable combination was overexpression of ponA combined with ltaS, which facilitated the AmyS activity (1.53-fold). Finally, the highest enzyme activity of recombinant strain reached 2901.6 U/mL. This study provided many manipulated targets for improving the production of recombinant proteins and laid foundations for functional annotation of genes in B. subtilis.
4-Hydroxyvaleric acid (4-HV) holds promise as a sustainable monomer for biodegradable polyesters and liquid transportation fuels. This study achieved high-level 4-HV production from levulinic acid using an antibiotic-free, substrate-inducible system in Escherichia coli. Enzymes involved in the conversion of levulinic acid to 4-HV were expressed with a bicistronic design of ribosome binding sites. The engineered strain demonstrated a 28% higher productivity compared to its counterpart, reaching a significant concentration of 107 g/L 4-HV with a production rate of 4.5 g/L/h and a molar conversion of 95% from levulinic acid in fed-batch cultivation. Recombinant cells from the initial cultivation were reused for a second round of biotransformation, demonstrating 73% efficiency of fresh cells. The study identified specific factors contributing to decreased system efficiency, including medium conditions, increased ionic strength, and high product concentration. Overall, the reported system and our findings hold significant potential for cost-effective microbial production of 4-HV at scale from levulinic acid.
This study highlights the production and characterization of siderophore from probiotic bacteria, aiming to evaluate its suitability as a carrier to treat iron deficiency anemia. Organisms were previously isolated from Chhurpi (Himalayan traditional fermented food) and their probiotic characteristics were reported. Among the, Bacillus subtilis L9 that produced catecholate and Pediococcus pentosaceus BAC L7 synthesized hydroxymate and catecholate type of siderophore. After optimization (pH 6.0, 100 rpm agitation speed and 15% inoculums volume) B. subtilis L9 and P. pentosaceus BAC L7 produced 89 and 73% siderophore unit respectively, after 72 h of incubation at simulated gastro-intestinal condition. Owing to better siderophore production and spore forming capability B. subtilis L9 was selected and physico-chemical characteristics of its siderophore were studied by FTIR, NMR and mass spectrometric analysis. The purified siderophore (2,3-dihydroxybenzoyl glycine) from B. subtilis L9 exhibited a high iron scavenging activity (89.76%). The study was penlights the exploration of probiotic microorganism present in the indigenous fermented food and their capability to biosynthesize siderophore that could be an effective therapeutic aid for maximum absorption of dietary iron and alleviation anemia like micronutrient deficiency diseases as well as to combat hypoxia among the high altitude residents.
The integration of sustainable technologies for energy generation in soybean biorefineries can enhance the bioeconomic performance of the production chain through the valorisation of agro-industrial residues. The aim of this work was to prepare a technological map of soybean ethanol technologies, forecasting the future of specific clusters related to ethanol production from soybean molasses, soybean lignocellulosic residues, and the use of soybean as a nitrogen source in alcoholic fermentations. A patent search was carried out to build a database of publication year, assignees, countries of origin and deposit, international patent classification codes and citations. United States and China were the most relevant countries contributing to patent applications, and 68% of the total documents were international deposits filed in multiple countries; 74% of the patent documents were filed with the participation of the industrial sector, the most frequent assignees being Du Pont, Iogen, and Mascoma. Technologies related to the use of whole soybean or molasses as carbon sources, or the use as a nitrogen source in ethanol fermentation, were found to be in their growing phases, while the use of soybean’s lignocellulosic fraction had already reached maturity stage. The highest values of patent power (12.23), expansion potential (297), and technology diffusion speed (19.70) were obtained for the cluster related to the exploitation of soybean’s lignocellulosic fraction. The conversion of soybean molasses to ethanol or the use of specific soybean components, including nitrogen sources, to improve alcoholic fermentation represents a more specific technology cluster that is gaining prominence in the industrial sector.
This study evaluates the encapsulation of phytoactives from acerola (Malpighia emarginata) pomace extract (APE) into Saccharomyces cerevisiae cells via sonoprocessing coupled to spray drying (SPSD process). The effect of acoustic energy density (AED; 0–333.3 W/L), APE pH (3–11), and spray drying inlet temperature (T; 150–190 °C) on the encapsulation efficiency (EE) and yield (EY) of phenolic compounds and anthocyanins were investigated through a Box–Behnken experimental design (BBD). Results demonstrated that maximized EEs (> 63%) and EYs (> 126.7 mg/100 g) were obtained using the highest AED (333.3 W/L) and alkaline APE (pH 11), while the optimal T was set at 170 °C. Likewise, these process conditions also enhanced the antioxidant activity of biocapsules which positively correlated with the concentration of yeast encapsulated phytoactives by SPSD. Overall, our results demonstrate the versatility of SPSD protocol for manufacturing bioactive-enriched yeast biocapsules using complex mixtures of phytoactives. Further, it presents an innovative and sustainable roadmap for the management and repurposing of agri-food byproducts such as acerola pomace and other secondary streams of the food industry.